Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus-specific cytotoxic T lymphocytes: Requirement for L3T4(+) T cells

The protective ability of cytotoxic T cells (CTL) raised in vitro against Japanese encephalitis virus (JEV) was examined by adoptive transfer experiments. Adoptive transfer of anti-JEV effecters by intracerebral (i.c.) but not by intraperitoneal (i.p.) or intravenous (i.v.) routes protected adult BALB/c mice against lethal i.c. JEV challenge. In contrast to adult mice, adoptive transfer of anti-JEV effecters into newborn (4-day-old) and suckling (8-14-day-old) mice did not confer protection. However, virus-induced death was delayed in suckling mice compared to newborn mice upon adoptive transfer. The specific reasons for lack of protection in newborn mice are not clear but virus load was found to be higher in newborn mice brains compared to those of adults and virus clearance was observed only in adult mice brains but not in newborn mice brains upon adoptive transfer. Specific depletion of Lyt 2.2(+), L3T4(+) or Thy-1(+) T cell populations before adoptive transfer abrogated the protective ability of transferred effecters. However, when Lyt 2.2(+) cell-depleted and L3T4(+) cell-depleted effecters were mixed and transferred into adult mice the protective activity was retained, demonstrating that both Lyt 2.2(+) and L3T4(+) T cells are necessary to confer protection. Although the presence of L3T4(+) T cells in adoptively transferred effector populations enhanced virus-specific serum neutralizing antibodies, the presence of neutralizing antibodies alone without Lyt 2.2(+) cells was not sufficient to confer protection.

Designed Cyclic Permutants of HIV-1 gp120: Implications for Envelope Trimer Structure and Immunogen Design

Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the env surface protein. Native env consists of a trimer of gp120-gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs)-directed neutralizing antibodies over non-neutralizing ones. Some cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface. We have therefore designed cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cycl, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with similar to 20-fold higher affinity than wild-type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support env models in which the V1V2 loops are proximal to the trimer interface.

A novel adeno-associated virus serotype (AAV)-8 capsid mutant improves human coagulation factor IX expression in vivo

Recombinant AAV-8 vectors have shown significant promise for hepatic gene therapy of hemophilia B. However, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. We hypothesized that AAV8 during its intracellular trafficking, are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery and modification of specific serine/threonine kinase or ubiquitination targets on AAV8 capsid (Fig.1A) may improve its transduction efficiency. To test this, point mutations at specific serine (S)/threonine (T) > alanine (A) or lysine (K)>arginine (R) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant(S276A, S501A, S671A, T251A and K137R) capsids were evaluated for their liver transduction efficiency at a dose of 5 X 1010 vgs/ animal in C57BL/6 mice in vivo. The best performing mutant was found to be the K137R vector in terms of either the gene expression (46-fold) or the vector copy numbers in the hepatocytes (22-fold) compared to WT-AAV8 (Fig.1B). The K137R-AAV8 vector that showed significantly decreased ubiquitination of the viral capsid had reduced activation of markers of innate immune response [IL-6, IL-12, tumor necrosis factor α, Kupffer cells and TLR-9]. In addition, animals injected with the K137R mutant also demonstrated decreased (2-fold) levels of cross-neutralizing antibodies when compared to animals that received the WT-AAV8 vector. To study further the utility of the novel AAV8-K137R mutant in a therapeutic setting, we delivered human coagulation factor IX (h.FIX) under the control of liver specific promoters (LP1 or hAAT) at two different doses (2.5x10^10 and 1x10^11 vgs per mouse) in 8-12 weeks old male C57BL/6 mice. As can be seen in Fig.1C/D, the circulating levels of h.FIX were higher in all the K137R-AAV8 treated groups as compared to the WT-AAV8 treated groups either at 2 weeks (62% vs 37% for hAAT constructs and 47% vs 21% for LP1 constructs) or 4 weeks (78% vs 56% for hAAT constructs and 64% vs 30% for LP1 constructs) post hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel vector for potential gene therapy of hemophilia B.

In vitro and in vivo characterization of designed immunogens derived from the CD-helix of the stem of influenza hemagglutinin

The conserved stem domain of influenza virus hemagglutinin (HA) is a target for broadly neutralizing antibodies and a potential vaccine antigen for induction of hetero-subtypic protection. The epitope of 12D1, a previously reported bnAb neutralizing several H3 subtype influenza strains, was putatively mapped to residues 76-106 of the CD-helix, also referred to as long alpha helix (LAH) of the HA stem. A peptide derivative consisting of wt-LAH residues 76-130 conjugated to keyhole limpet hemocyanin was previously shown to confer robust protection in mice against challenge with influenza strains of subtypes H3, H1, and H5 which motivated the present study. We report the design of multiple peptide derivatives of LAH with or without heterologous trimerization sequences and show that several of these are better folded than wt-LAH. However, in contrast to the previous study immunization of mice with wt-LAH resulted in negligible protection against a lethal homologous virus challenge, while some of the newly designed immunogens could confer weak protection. Combined with structural analysis of HA, our data suggest that in addition to LAH, other regions of HA are likely to significantly contribute to the epitope for 12D1 and will be required to elicit robust protection. In addition, a dynamic, flexible conformation of isolated LAH peptide may be required for eliciting a functional anti-viral response. Proteins 2013; 81:1759-1775. (c) 2013 Wiley Periodicals, Inc.

Immunogen design for HIV-1 and influenza

Vaccines provide the most cost effective defense against pathogens. Although vaccines have been designed for a number of viral diseases, a vaccine against HIV-1 still remains elusive. In contrast while there are excellent influenza vaccines, these need to be changed every few years because of antigenic drift and shift The recent discovery of a large number of broadly neutralizing antibodies (bNAbs) and structural characterization of the conserved epitopes targeted by them presents an opportunity for structure based HIV-1 and influenza A vaccine design. We discuss strategies to design immunogens either targeting a particular antigenic region or focusing on native structure stabilization. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. (C) 2014 Elsevier B.V. All rights reserved.

Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy

Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy. (C) 2015 Elsevier Ltd. All rights reserved.

Stalking influenza by vaccination with pre-fusion headless HA mini-stem

Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies indudced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity.

Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2

Background: Human melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown. Methods: Herein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied. Results: Mice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNF alpha and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion-and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFa induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1. Conclusions: We demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion-and proliferation-stimulating effects of TNFa, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing

Robust Control of Evolutionary Dynamics

The application of principles from evolutionary biology has long been used to gain new insights into the progression and clinical control of both infectious diseases and neoplasms. This iterative evolutionary process consists of expansion, diversification and selection within an adaptive landscape - species are subject to random genetic or epigenetic alterations that result in variations; genetic information is inherited through asexual reproduction and strong selective pressures such as therapeutic intervention can lead to the adaptation and expansion of resistant variants. These principles lie at the center of modern evolutionary synthesis and constitute the primary reasons for the development of resistance and therapeutic failure, but also provide a framework that allows for more effective control.

A model system for studying the evolution of resistance and control of therapeutic failure is the treatment of chronic HIV-1 infection by broadly neutralizing antibody (bNAb) therapy. A relatively recent discovery is that a minority of HIV-infected individuals can produce broadly neutralizing antibodies, that is, antibodies that inhibit infection by many strains of HIV. Passive transfer of human antibodies for the prevention and treatment of HIV-1 infection is increasingly being considered as an alternative to a conventional vaccine. However, recent evolution studies have uncovered that antibody treatment can exert selective pressure on virus that results in the rapid evolution of resistance. In certain cases, complete resistance to an antibody is conferred with a single amino acid substitution on the viral envelope of HIV.

The challenges in uncovering resistance mechanisms and designing effective combination strategies to control evolutionary processes and prevent therapeutic failure apply more broadly. We are motivated by two questions: Can we predict the evolution to resistance by characterizing genetic alterations that contribute to modified phenotypic fitness? Given an evolutionary landscape and a set of candidate therapies, can we computationally synthesize treatment strategies that control evolution to resistance?

To address the first question, we propose a mathematical framework to reason about evolutionary dynamics of HIV from computationally derived Gibbs energy fitness landscapes -- expanding the theoretical concept of an evolutionary landscape originally conceived by Sewall Wright to a computable, quantifiable, multidimensional, structurally defined fitness surface upon which to study complex HIV evolutionary outcomes.

To design combination treatment strategies that control evolution to resistance, we propose a methodology that solves for optimal combinations and concentrations of candidate therapies, and allows for the ability to quantifiably explore tradeoffs in treatment design, such as limiting the number of candidate therapies in the combination, dosage constraints and robustness to error. Our algorithm is based on the application of recent results in optimal control to an HIV evolutionary dynamics model and is constructed from experimentally derived antibody resistant phenotypes and their single antibody pharmacodynamics. This method represents a first step towards integrating principled engineering techniques with an experimentally based mathematical model in the rational design of combination treatment strategies and offers predictive understanding of the effects of combination therapies of evolutionary dynamics and resistance of HIV. Preliminary in vitro studies suggest that the combination antibody therapies predicted by our algorithm can neutralize heterogeneous viral populations despite containing resistant mutations.

Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

Comparative immunogenicity of HIV-1 clade C envelope proteins for prime/boost studies.

BACKGROUND: Previous clinical efficacy trials failed to support the continued development of recombinant gp120 (rgp120) as a candidate HIV vaccine. However, the recent RV144 HIV vaccine trial in Thailand showed that a prime/boost immunization strategy involving priming with canarypox vCP1521 followed by boosting with rgp120 could provide significant, although modest, protection from HIV infection. Based on these results, there is renewed interest in the development of rgp120 based antigens for follow up vaccine trials, where this immunization approach can be applied to other cohorts at high risk for HIV infection. Of particular interest are cohorts in Africa, India, and China that are infected with clade C viruses. METHODOLOGY/PRINCIPAL FINDINGS: A panel of 10 clade C rgp120 envelope proteins was expressed in 293 cells, purified by immunoaffinity chromatography, and used to immunize guinea pigs. The resulting sera were collected and analyzed in checkerboard experiments for rgp120 binding, V3 peptide binding, and CD4 blocking activity. Virus neutralization studies were carried out with two different assays and two different panels of clade C viruses. A high degree of cross reactivity against clade C and clade B viruses and viral proteins was observed. Most, but not all of the immunogens tested elicited antibodies that neutralized tier 1 clade B viruses, and some sera neutralized multiple clade C viruses. Immunization with rgp120 from the CN97001 strain of HIV appeared to elicit higher cross neutralizing antibody titers than the other antigens tested. CONCLUSIONS/SIGNIFICANCE: While all of the clade C antigens tested were immunogenic, some were more effective than others in eliciting virus neutralizing antibodies. Neutralization titers did not correlate with rgp120 binding, V3 peptide binding, or CD4 blocking activity. CN97001 rgp120 elicited the highest level of neutralizing antibodies, and should be considered for further HIV vaccine development studies.

Safety and immunogenicity of a replication-defective adenovirus type 5 HIV vaccine in Ad5-seronegative persons: a randomized clinical trial (HVTN 054).

BACKGROUND: Individuals without prior immunity to a vaccine vector may be more sensitive to reactions following injection, but may also show optimal immune responses to vaccine antigens. To assess safety and maximal tolerated dose of an adenoviral vaccine vector in volunteers without prior immunity, we evaluated a recombinant replication-defective adenovirus type 5 (rAd5) vaccine expressing HIV-1 Gag, Pol, and multiclade Env proteins, VRC-HIVADV014-00-VP, in a randomized, double-blind, dose-escalation, multicenter trial (HVTN study 054) in HIV-1-seronegative participants without detectable neutralizing antibodies (nAb) to the vector. As secondary outcomes, we also assessed T-cell and antibody responses. METHODOLOGY/PRINCIPAL FINDINGS: Volunteers received one dose of vaccine at either 10(10) or 10(11) adenovector particle units, or placebo. T-cell responses were measured against pools of global potential T-cell epitope peptides. HIV-1 binding and neutralizing antibodies were assessed. Systemic reactogenicity was greater at the higher dose, but the vaccine was well tolerated at both doses. Although no HIV infections occurred, commercial diagnostic assays were positive in 87% of vaccinees one year after vaccination. More than 85% of vaccinees developed HIV-1-specific T-cell responses detected by IFN-γ ELISpot and ICS assays at day 28. T-cell responses were: CD8-biased; evenly distributed across the three HIV-1 antigens; not substantially increased at the higher dose; and detected at similar frequencies one year following injection. The vaccine induced binding antibodies against at least one HIV-1 Env antigen in all recipients. CONCLUSIONS/SIGNIFICANCE: This vaccine appeared safe and was highly immunogenic following a single dose in human volunteers without prior nAb against the vector. TRIAL REGISTRATION: ClinicalTrials.gov NCT00119873.

A PK2/Bv8/PROK2 antagonist suppresses tumorigenic processes by inhibiting angiogenesis in glioma and blocking myeloid cell infiltration in pancreatic cancer.

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy.

The Ontogeny of Mucosal and Systemic Antibody Responses to HIV-1 Infection

The humoral immune system plays a critical role in the clearance of numerous pathogens. In the setting of HIV-1 infection, the virus infects, integrates its genome into the host's cells, replicates, and establishes a reservoir of virus-infected cells. The initial antibody response to HIV-1 infection is targeted to non-neutralizing epitopes on HIV-1 Env gp41, and when a neutralizing response does develop months after transmission, it is specific for the autologous founder virus and the virus escapes rapidly. After continuous waves of antibody mediated neutralization and viral escape, a small subset of infected individuals eventually develop broad and potent heterologous neutralizing antibodies years after infection. In this dissertation, I have studied the ontogeny of mucosal and systemic antibody responses to HIV-1 infection by means of three distinct aims: 1. Determine the origin of the initial antibody response to HIV-1 infection. 2. Characterize the role of restricted VH and VL gene segment usage in shaping the antibody response to HIV-1 infection. 3. Determine the role of persistence of B cell clonal lineages in shaping the mutation frequencies of HIV-1 reactive antibodies.

After the introduction (Chapter 1) and methods (Chapter 2), Chapter 3 of this dissertation describes a study of the antibody response of terminal ileum B cells to HIV-1 envelope (Env) in early and chronic HIV-1 infection and provides evidence for the role of environmental antigens in shaping the repertoire of B cells that respond to HIV-1 infection. Previous work by Liao et al. demonstrated that the initial plasma cell response in the blood to acute HIV-1 infection is to gp41 and is derived from a polyreactive memory B cell pool. Many of these antibodies cross-reacted with commensal bacteria, Therefore, in Chapter 3, the relationship of intestinal B cell reactivity with commensal bacteria to HIV-1 infection-induced antibody response was probed using single B cell sorting, reverse transcription and nested polymerase chain reaction (RT- PCR) methods, and recombinant antibody technology. The dominant B cell response in the terminal ileum was to HIV-1 envelope (Env) gp41, and 82% of gp41- reactive antibodies cross-reacted with commensal bacteria whole cell lysates. Pyrosequencing of blood B cells revealed HIV-1 antibody clonal lineages shared between ileum and blood. Mutated IgG antibodies cross-reactive with both Env gp41 and commensal bacteria could also be isolated from the terminal ileum of HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 can be shaped by intestinal B cells stimulated by commensal bacteria prior to HIV-1 infection to develop a pre-infection pool of memory B cells cross-reactive with HIV-1 gp41.

Chapter 4 details the study of restricted VH and VL gene segment usage for gp41 and gp120 antibody induction following acute HIV-1 infection; mutations in gp41 lead to virus enhanced neutralization sensitivity. The B cell repertoire of antibodies induced in a HIV-1 infected African individual, CAP206, who developed broadly neutralizing antibodies (bnAbs) directed to the HIV-1 envelope gp41 membrane proximal external region (MPER), is characterized. Understanding the selection of virus mutants by neutralizing antibodies is critical to understanding the role of antibodies in control of HIV-1 replication and prevention from HIV-1 infection. Previously, an MPER neutralizing antibody, CAP206-CH12, with the binding footprint identical to that of MPER broadly neutralizing antibody 4E10, that like 4E10 utilized the VH1-69 and VK3-20 variable gene segments was isolated from this individual (Morris et al., 2011). Using single B cell sorting, RT- PCR methods, and recombinant antibody technology, Chapter 4 describes the isolation of a VH1-69, Vk3-20 glycan-dependent clonal lineage from CAP206, targeted to gp120, that has the property of neutralizing a neutralization sensitive CAP206 transmitted/founder (T/F) and heterologous viruses with mutations at amino acids 680 or 681 in the MPER 4E10/CH12 binding site. These data demonstrate sites within the MPER bnAb epitope (aa 680-681) in which mutations can be selected that lead to viruses with enhanced sensitivity to autologous and heterologous neutralizing antibodies.

In Chapter 5, I have completed a comparison of evolution of B cell clonal lineages in two HIV-1 infected individuals who have a predominant VH1-69 response to HIV-1 infection--one who produces broadly neutralizing MPER-reactive mAbs and one who does not. Autologous neutralization in the plasma takes ~12 weeks to develop (Gray et al., 2007; Tomaras et al., 2008b). Only a small subset of HIV-1 infected individuals develops high plasma levels of broad and potent heterologous neutralization, and when it does occur, it typically takes 3-4 years to develop (Euler et al., 2010; Gray et al., 2007; 2011; Tomaras et al., 2011). The HIV-1 bnAbs that have been isolated to date have a number of unusual characteristics including, autoreactivity and high levels of somatic hypermutations, which are typically tightly regulated by immune control mechanisms (Haynes et al., 2005; 2012b; Kwong and Mascola, 2012; Scheid et al., 2009a). The VH mutation frequencies of bnAbs average ~15% but have been shown to be as high as 32% (reviewed in Mascola and Haynes, 2013; Kwong and Mascola, 2012). The high frequency of somatic hypermutations suggests that the B cell clonal lineages that eventually produce bnAbs undergo high-levels of affinity maturation, implying prolonged germinal center (GC) reactions and high levels of T cell help. To study the duration of HIV-1- reactive B cell clonal persistence, HIV-1 reactive and non HIV-1- reactive B cell clonal lineages were isolated from an HIV-1 infected individual that produces bnAbs, CAP206, and an HIV-1 infected individual who does not produce bnAbs, 004-0. Single B cell sorting, RT-PCR and recombinant antibody technology was used to isolate and produce monoclonal antibodies from multiple time points from each individual. B cell sequences clonally related to mAbs isolated by single cell PCR were identified within pyrosequences of longitudinal samples of these two individuals. Both individuals produced long-lived B cell clones that persisted from 0-232 weeks in CAP206, and 0-238 weeks in 004-0. The average length of persistence of clones containing members isolated from two separate time points was 91.5 weeks both individuals. Examples of the continued evolution of clonal lineages were observed in both the bnAb and non-bnAb individual. These data indicated that the ability to generate persistent and evolving B cell clonal lineages occurs in both bnAb and non-bnAb individuals, suggesting that some alternative host or viral factor is critical for the generation of highly mutated broadly neutralizing antibodies.

Together the studies described in Chapter 3-5 show that multiple factors influence the antibody response to HIV-1 infection. The initial antibody response to HIV-1 Env gp41 can be shaped by a B cell response to intestinal commensal bacteria prior to HIV-1 infection. VH and VL gene segment restriction can impact the B cell response to multiple HIV-1 antigens, and virus escape mutations in the MPER can confer enhanced neutralization sensitivity to autologous and heterologous antibodies. Finally, the ability to generate long-lived HIV-1 clonal lineages in and of itself does not confer on the host the ability to produce bnAbs.

Infection of monkeys by simian-human immunodeficiency viruses with transmitted/founder clade C HIV-1 envelopes.

Simian-human immunodeficiency viruses (SHIVs) that mirror natural transmitted/founder (T/F) viruses in man are needed for evaluation of HIV-1 vaccine candidates in nonhuman primates. Currently available SHIVs contain HIV-1 env genes from chronically-infected individuals and do not reflect the characteristics of biologically relevant HIV-1 strains that mediate human transmission. We chose to develop clade C SHIVs, as clade C is the major infecting subtype of HIV-1 in the world. We constructed 10 clade C SHIVs expressing Env proteins from T/F viruses. Three of these ten clade C SHIVs (SHIV KB9 C3, SHIV KB9 C4 and SHIV KB9 C5) replicated in naïve rhesus monkeys. These three SHIVs are mucosally transmissible and are neutralized by sCD4 and several HIV-1 broadly neutralizing antibodies. However, like natural T/F viruses, they exhibit low Env reactivity and a Tier 2 neutralization sensitivity. Of note, none of the clade C T/F SHIVs elicited detectable autologous neutralizing antibodies in the infected monkeys, even though antibodies that neutralized a heterologous Tier 1 HIV-1 were generated. Challenge with these three new clade C SHIVs will provide biologically relevant tests for vaccine protection in rhesus macaques.